The Morphology of Trichomes
There are two types of trichomes on the cotton leaf surface, glandular trichomes and covering pubescences (asteroid non-glandular trichomes), observed with scanning electron microscopy (Fig. 1G). Asteroid pubescences are distributed along the leaf veins, and the branches of asteroid pubescence are mainly ranged from 1 to 6 (Fig. 1A, B, C and D). While glandular trichomes, widely distributed on the paraxial and abaxial surfaces of leaves, are short and thick and have an 8-cell head and a stalk (Fig. 1E and F).
EPG Recording and Analysis
A complete feeding process could be divided into three periods according to the sequential occurrence of different waveforms: Period I, II, and III. Period I lasted from the start of probing to the first phloem waveform and was characterized by testing on the leaf surface, tasting intracell, and starting sucking from the phloem. During the test stage, aphids were searching for suitable penetration sites. The inactive phase without penetration is referred to as the non-probing phase named np waveform. The stylet penetration phase encompasses brief intercellular punctures by which the insect attempts to locate its piercing site or makes the decision on host selection, and the most waveform was the pathway waveform C. Intracellular advancement and withdrawal penetration will be recorded as potential drop (pd) waveform. From the beginning of the first phloem-sucking waveform E, the phloem taste stage (Period II) started, and the continuous feeding stage (Period III) was from the first E waveform with a duration beyond 10 minutes. In the last stage, the pathway waveform C and the phloem-sucking waveform E appeared alternately.
Among the selected EPG parameters related to the feeding behaviors of aphids on those three types of cotton plants, 2 parameters were found significantly different (P<0.05), and 1 parameter was found extremely significant difference (P<0.01), while there were 6 parameters being marginally significant different (0.05<P<0.10). However, several parameters between every two treatments were significantly different, which could show the consequences of treatment on the trichomes.
General Parameters in EPG
The general performance of aphids could be described with twelve non-sequential EPG parameters (Table 1). The parameter “total number of potential drops (pds)” of treatment DF was found to be the highest among the three treatments, and significantly more than that of treatment TR (P<0.05), indicating that aphids punctured more times into tissue cells and tasted the cytoplasm on DF leaves.
Period I (from the beginning of recording to the first phloem waveform E)
There were 13 parameters, both sequential and non-sequential, that reflected the searching behavior for confirming suitable penetration sites and assessed the performance of aphids during the test stage (Table 2). This stage ended with the beginning of the first waveform E. EPG records showed no significant differences between TR and MD, which indicated that treatments had tiny effects on the total puncture behaviors of aphids. The parameters including the time of the first probe were extremely earlier and the np duration in Period I was extremely shorter in DF (P<0.01), while there were no significant differences between TR and MD. The rest of the parameters showed no significant difference. The above-mentioned parameters demonstrated that the aphids spent a longer time searching the penetrating site on the treated leaves, and the barrier in the mesophyll was similar once the penetrating site was chosen.
Period II (from the first phloem waveform E to the first sustained phloem ingestion)
During the mesophyll-sucking stage, the aphid secreted saliva and solidified around the stylet as a salivary sheath by which the aphid explored the epidermis and mesophyll cells. EPG showed a C waveform with large fluctuation and potential drop waveform. The E waveforms longer than 10min were recognized as sustained phloem ingestion.
In Period II (Table 3), the aphids on the MD leaves spent more time on the 1st sustained phloem ingestion, mechanical damage significantly delayed the first phloem-feeding (P<0.5). For other indicators, compared with TR and MD, the duration of the C waveform before the first sustained ingestion was longer, the duration of the C waveform in Period II was shorter, and the number of pds in the pathway before the first sustained ingestion was more in the leaf with trichomes. These parameters showed no significant difference, but they tended to indicate that it’s more difficult for aphids to penetrate the mesophyll cells and conduct phloem-sucking in normal cotton leaves.
Period III (from the first sustained ingestion to the end of recording)
When the stylet reached the phloem, aphid began to ingest sap passively, and EPG was recorded as an E waveform with negative voltage. There was only one parameter, the mean number of pds in the pathway following sustained phloem ingestion, with significant differences in that period among three treatments, while more and longer np and C waveforms were shown on damage-free leaves (Table 4). Combined with Period II (Table 4) we can find that phloem-sucking was earlier but longer in damaged-free leaves, and aphids on intact leaves penetrated the mesophyll and changed the penetrated sites more times to gain similar phloem-feeding.